In modern medicine, cellular therapies, regenerative medicine and tissue engineering all involve technologies for harvesting, expanding, modifying and re-implanting live viable cells and tissues. A primary example is the transplantation of isolated pancreatic islets of Langerhans for the treatment of Type I (insulin dependent) diabetes. Ever since the first experimental attempts to ameliorate Type I diabetes by transplantation of allograft donor islets the field has been challenged by the need for improved methods of retrieving islets from donor pancreata. In fact, there is a considerable worldwide effort to further develop the concept for treating Type I diabetes by transplanting islets, but clinical application of the techniques developed in animal models is fraught with many challenges. The field of islet transplantation generally relies upon enzymatic digestion processes that destroys the extracellular matrix of the tissue, releasing the entrapped islets for further processing and purification. This widely practiced procedure has drawbacks due principally to the difficulty of controlling the digestive process to yield an optimum quantity of viable cells.
The source of the islets also remains a primary concern, and isolation from donor pancreases demands resolution of questions concerning the source, supply, and condition of the donor organs. Reliance upon an adequate supply of human organs for this purpose is considered futile, such that alternative sources are actively been sought (Bonner-Weir, S. et al., New sources of pancreatic beta-cells, Nat. Biotechnol. 23:857-861, 2005; Hering, B. J. et al., Prolonged diabetes reversal after intraportal xenotransplantation of wild-type porcine islets in immunosuppressed nonhuman primates, Nat. Med., 12:301-303, 2006; Inada, A.; Bonner-Weir, S. et al., How can we get more beta cells?, Curr. Diab. Rep., 6:96-101, 2006).
Various mammals are considered optimal candidates for xenogeneic islet transplantation. Of the potential mammals, pigs are considered the donor species of choice for xenogeneic islet transplantation for a number of compelling reasons. Pigs share many physiological similarities to humans and porcine insulin has demonstrated clinical efficacy for many years. Pigs are raised as a food source and provide an ethical source of donor islets by being housed in a controlled environment to ensure safety for porcine islet xenotransplantation. However, experiences in many laboratories over the past 10 years show that isolation of porcine islets appears to be more difficult (Finke, E., et al., Large scale isolation, function, and transplantation of islets of Langerhans from the adult pig pancreas. Transplant. Proc. 23:772-773, 1991; Giannarelli, R. et al., Preparation of pure, viable porcine and bovine islets by a simple method. Transplant. Proc., 26:630-631, 1994; Marchetti, P. et al., Automated largescale isolation, in vitro function and xenotransplantation of porcine islets of Langerhans, Transplantation 52:209-213, 1991; O'Neil, J. J. et al., The isolation and function of porcine islets from market weight pigs. Cell Transplant., 10:235-246, 2001; Toso, C. et al., Isolation of adult porcine islets of Langerhans. Cell Transplant., 9:297-305, 2000), compared with the isolation of human (Kenmochi, T. et al., Improved quality and yield of islets isolated from human pancreas using two-step digestion method, Pancreas 20:184-190, 2000), bovine (Figliuzzi, M. et al., Influence of donor age on bovine pancreatic islet isolation, Transplantation, 70:1032-1037, 2000), or rodent islets (Shapiro, A. M. et al., High yield of rodent islets with intraductal collagenase and stationary digestion—a comparison with standard technique, Cell Transplant., 5:631-638, 1996).
Porcine islets are less compact and tend to fragment during the isolation procedure and during prolonged periods of in vitro culture (Ricordi, C. et al., A method for the mass isolation of islets from the adult pig pancreas, Diabetes, 35:649-653, 1986). Moreover, the age, and even the strain, of the donor pig has been documented by several groups to markedly influence the islet isolation process, with young, so-called market size pigs (<6 months old) proving to be particularly difficult as a source of transplantable islets (Bottino, R. et al., Isolation outcome and functional characteristics of young and adult pig pancreatic islets for transplantation studies, Xenotransplantation, 14:74-82, 2007; Dufrane, D. et al., Impact of porcine islet size on cellular structure and engraftment after transplantation: Adult versus young pigs, Pancreas 30:138-147, 2005; Toso, C. et al., Isolation of adult porcine islets of Langerhans. Cell Transplant., 9:297-305, 2000). Islets from adult pigs (>2 years old) offered not only higher yields, but retained the ability to preserve intact morphology during the isolation process and culture, in association with higher functional properties after transplantation. Despite the challenge encountered by many groups attempting to isolate islets from young pigs, donor pigs of market weight (<80 kg=<12 months old) are preferred to retired breeders (>200 kg=>2 years old) due to their abundance, lower animal and husbandry costs, and they are more suitable to meet regulatory guidelines for donor tissue for xenotransplantation. The methods of this disclosure may improve the cellular product yield from donor tissues and improve the efficacy of hypothermic machine perfusion (HMP) of donor tissues, such as pancreata, prior to use, such as during islet isolation.
The scientific basis for hypothermic perfusion preservation of organs is founded upon the effect of temperature on all biologic and chemical processes, which are fundamentally slowed by a reduction of temperature. Hence the deleterious consequences of ischemia and anoxia can be attenuated by the application of hypothermia, which has provided the cornerstone of most of the effective methods of organ preservation in common use today. Hypothermic perfusion preservation is based upon the fundamental premise that devices can be designed to facilitate the replacement of blood in the circulation of an ex vivo organ with specially designed fluids to maximize the protective effects of hypothermia on the ischemic tissue.
Since the advent of clinical organ transplantation in the 1960's, a variety of perfusion machines have been developed principally for kidney preservation, but until recently these were not employed clinically due to the relatively high cost and complexity compared with simple cold storage techniques. Today, there is a growing use of machine perfusion for donor kidney preservation due to the reported effect of improved outcome using so-called “marginal” or “expanded criteria” donor organs. This technique therefore has a major potential impact upon increasing the numbers of organs available for transplantation. One of the commercially available machines (LifePort®; LifeLine Scientific) approved for clinical use for kidneys may be utilized in the methods associated with the present application improving the cellular product yield from donor tissues either with or without hypothermic preservation.
Earlier studies have demonstrated that hypothermic preservation of organs, such as the pancreas, by machine perfusion is feasible and may be safely extended to 24 and 48 h (Alteveer, R. J. et al., Hemodynamics and metabolism of the in vivo vascularly isolated canine Pancreas, Am. J. Physiol., 236:E626-E632, 1979; Florack, G. et al., Preservation of canine segmental pancreatic autografts: Cold storage versus pulsatile machine perfusion, J. Surg. Res., 34:493-504, 1983; Leeser, D. B. et al., Pulsatile pump perfusion of pancreata before human islet cell isolation, Transplant, Proc. 36:1050-1051, 2004; Tersigni, R. et al., Pancreaticoduodenal preservation by hypothermic pulsatile perfusion for twenty-four hours, Ann. Surg., 182: 743-748, 1975; Toledo-Pereyra, L. H., Hypothermic pulsatile perfusion: Its use in the preservation of pancreases for 24 to 48 hours before islet cell transplantation, Arch. Surg., 115:95-98, 1980; Moers, C. et al., Machine perfusion or cold storage in deceased-donor kidney transplantation, N. Engl. J. Med., 360:7-19, 2009; Rakhorst, G. et al., Revival of machine perfusion: New chances to increase the donor pool? Expert Rev. Med. Devices 2:7-8, 2005; Reznik, O. N. et al., Increasing kidneys donor's pool by machine perfusion with the LifePort-pilot Russian study, Ann. Transplant, 11:46-48, 2006; Taylor, M. J. et al., Current state of hypothermic machine perfusion preservation of organs: The clinical perspective, Cryobiology, in press).
Dedicated renal perfusion systems may be employed by the methods of the present disclosure after appropriate modifications are made to accommodate the characteristics of the respective organ, such as, for example, the physiologic low flow and pressure needs of the pancreas. The latter helps avoid excessive organ edema that postsegmental transplantation and reperfusion has been documented to result in subcapsular bleeding, hemorrhagic necrosis, venous congestion, and hemorrhagic pancreaticoduodenal secretions.
Transplantation of cellular products has been previously reported. For example, transplanted islets isolated from 24-h perfused dog pancreata have been reported to result in 60% recipient survival post transplantation, providing similar outcome to fresh islets implantation. Islets isolated from human pancreas after 13 h of cold static storage and 4 h of hypothermic pulsatile perfusion on a Waters RM3 system were characterized by higher viable yield and stimulation index relative to cells isolated from organs that sustained more than 8 h of static storage alone (Gondolesi, G. E. et al., Reduction of ischemia-reperfusion injury in parenchymal and nonparenchymal liver cells by donor treatment with DL-alpha-tocopherol prior to organ harvest, Transplant. Proc., 34:1086-1091, 2002).
These studies clearly provide the basis for a major clinical/commercial impact for new technologies that provide desperately needed improved methods of pancreas preservation to produce better yields of high quality islets. Clearly, islet transplantation is emerging as a viable option for the treatment of insulin-dependent diabetes mellitus, and clinical trials are under way at many centers around the world (Alejandro, R. et al., 2008 update from the Collaborative Islet Transplant Registry, Transplantation 86:1783-1788, 2008; and Shapiro, A. M. et al., International trial of the Edmonton protocol for islet transplantation. N. Engl. J. Med. 355:1318-1330, 2006). Accordingly, the demand for donor islets is escalating and will continue to grow. Thus, there is a need for higher quality and quantities of islets.
Despite many efforts to improve the technique of islet isolation, the field remains constrained by the limitations and vagaries of enzymatic digestion of a gland that comprises less than 5% endocrine tissue. Consequently, harvesting islets from a single donor pancreas often yields insufficient islet mass to reverse diabetes in a recipient, such that multiple donors often have to be considered for treating a single recipient.
The potential for xenotransplantation to relieve the demand on an inadequate supply of human pancreases depends upon the efficiency of techniques for isolating islets from the source pancreases (Hering, B. J. et al., The International Xenotransplantation Association consensus statement on conditions for undertaking clinical trials of porcine islet products in type 1 diabetes—executive summary, Xenotransplantation 16:196-202, 2009). However, at this time, procurement of donor pancreases for islet isolation and transplantation is not yet widely practiced due in part to concerns about postmortem ischemia upon functional islet yields.